Study And Application Of Genechip Technology On New Enteric Pathogens Detection

The traditional methods of bacteria culture and PCR technology can no longer meet the demand to make a fast detection for a pathogen in a level of great quantity. The development of DNA chips (microarray) makes available a powerful, high throughput detection and screening method for diagnosis,which is thousands and hundreds of times more efficient than the traditional ones. However,the research of applying microarray technology to pathogen detection is still at the primary stage. In the present study,the stx1, stx2 toxin gene and uidA mutation gene from Escherichia coli O157:H7 and the ctxA, tcpA toxin gene and LPSgt mutation gene from cholerae O139 were selected as main research materials to study the microarray technology, which used in the Escherichia coli O157:H7 and cholerae O139 detection and identification research,and the following key points and problems were studied and discussed. Ⅰ. It was proved from the study on the fabrication of the oligonucleotide microarray that the best way to preparation of the microarray for diagnosis of the enteric pathogens was to spot oligonucleotide probes with the amine modifications onto the aldehyde slides. Ⅱ. A PCR method was established and stx1, stx2, uidA, ctxA, tcpA and LPSgt gene comes from Escherichia coli O157:H7 and cholerae O139 respectively were amplified and sequenced. Results showed that the amplified fragments were the same as the expected one in size and the sequence of every cloned target DNA fragment no difference from the fragment which amplified by its primers. Ⅲ. Two set of multiplexed PCR system were established and optimized. It was proved that the amplified products were optimal in efficiency and equation. Ⅳ. Only signals which produced by six probes selected from sixty synthesized probes for Escherichia coli O157:H7 and cholerae O139 were same as the expected results. The specificity and sensitivity related to the six capture probes were the most satisfactory, they can be used to detect both Escherichia coli O157:H7 and cholerae O139 pathogens. Ⅴ . Some factors affecting multiplex PCR products hybridization signals were optimized and compared with the common hybridization system. The results showed that the optimal hybridization time and temperature were 60 minutes and 50 ℃ respectively, formamide concentration was 2.5%, with the other factors being the same as the general condition. Denaturalization of labeled PCR products should be emphasized before hybridization. Ⅵ. The results of the whole study showed that the gene chip probes were reasonablly designed and prepared, every probe was characterized by specificity. The sensitivity of those chips reached 100 copies DNA templates in one PCR system, 10 times more efficient than the common PCR, and has a higher sensitivity compared with the bacteriology clinical detection method. The results of the present study have made some helpful exploration and research in applying oligonucleotide microarray to Escherichia coli O157:H7 and cholerae O139 detection and has paved the road to make higher density oligonucleotide microarray to detect and identify more pathogens.